The overall objective of the proposed research is to explore the biosynthetic incorporation of galactofuranose (Galf) residues. Galf residues are found in many pathogens, but they are not present in mammals. In this application, we focus on understanding and inhibiting two enzymes involved in the biosynthesis of glycoconjugates containing Galf residues: the flavoenzyme UDP-galactopyranose mutase (UGM) and the putative galactofuranosyltransferase GlfT. Both of these enzymes are involved in mycobacterial cell wall biosynthesis, and both are essential for the viability of Mycobacterium tuberculosis, the causative agent of tuberculosis. Despite their potential importance as therapeutic targets, many questions regarding how these enzymes participate in biosynthesis of the mycobacterial cell wall remain. For example, the mechanism by which UGM catalyzes the isomerization of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf) is unknown. Even less is known about GlfT, which appears to transfer galactofuranose residues to lipid precursors. The proposed research has three aims. The first aim is focused on testing our hypothesis that the isomerization of UDP- Galp and UDP-Galf proceeds via an N(5) flavin-derived iminium ion. This mode of flavin catalysis has not been observed previously/and the experiments are proposed in aim 1 are designed to evaluate the feasibility of this mechanistic proposal. Aim 2 is directed at identifying inhibitors of UGM using a fluorescence polarization assay. One goal of this aim is to identify molecular scaffolds from which combinatorial libraries can be generated to optimize potency. In aim 3, we propose to investigate GlfT. The goals are to develop an effective method to produce this putative glycosyltransferase and to characterize its substrate specificity. We anticipate that the studies proposed here will open new avenues for investigating the roles of this interesting carbohydrate unit. Moreover, we hope that by pursuing the proposed investigations, new leads for the treatment of tuberculosis may emerge.